In the biflagellate alga Chlamydomonas, flagellar detachment signals the immediate and coordinate upregulation of over 150 genes encoding flagellar proteins. Synthesis of flagellar proteins rapidly ensues and, within minutes, new flagellar assembly is initiated. To understand the process of organelle assembly we will study the synthesis and assembly of the flagellar radical spokes. These structures, found only in the flagella, are required for flagellar motility; they are attached to the A-tubules of the outer doublet microtubules and are composed of 17 radial spoke polypeptides (RSPs) assembled into separable head and stalk domains. We have cloned and sequenced the genes for several of these polypeptides, successfully using them to rescue mutants defective in spoke assembly. Chlamydomonas is a polarized cell with the flagella at one end. With RSP antibodies and RSP cDNAs, we will determine by immunoelectron microscopy and in situ hybridization the site of RSP synthesis in the cytoplasm and subsequent route to the flagella. We will also determine if the 17 RSPs form cytoplasmic pre-assembly aggregates prior to their transport to the flagellum. To examine the assembly of spoke proteins onto the axoneme, RSPs obtained from cytoplasmic and flagellar extracts will be assembled onto axonemes isolated from mutants lacking entire spokes or spoke heads. We will continue to characterize the axonemal binding domain of RSP3 in vivo by testing the ability of mutagenized RSP3 to enter the flagellum, bind the axoneme, and target fusion proteins to the flagellar compartment. To determine how the RSPs and other flagellar proteins are transported to their assembly site at the flagellar tip, we will identify the molecular motors responsible for a newly-described motility beneath the flagellar membrane, IFT (Intraflagellar Transport). In addition, we will use biochemical and genetic procedures, including our probes for RSPs and other flagellar proteins, to determine the nature of the cargo being carried by these motors. While investigating motors that may power IFT, we have determined that a kinesin-like protein (Klpl), the cDNA of which we have cloned and sequenced, is localized to only one of the two central pair microtubules of the flagellar axoneme. We will clone and sequence the other kinesins we have identified in Chlamydomonas flagella and localize these proteins with specific antibody probes. Since the central-pair microtubules are thought to rotate during flagellar beating, we will examine Klpl motility in vitro assays, testing for directionality and the ability to rotate microtubules. Proteins that interact with Klpl will be identified by non-denaturing immunoprecipitation with the Klpl antibody. In addition, Klpl cDNAs will be mutagenized in vitro, and expressed in Chlamydomonas, to determine the functional role of this kinesin in vivo.